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In the first study to count mitochondrial genes in single motor neurons, researchers have found that motor neurons from people who had died of amyotrophic lateral sclerosis are likely to have mitochondrial DNA deficiencies and deletions. These mitochondrial problems might then lead to neurodegeneration, said senior author James Bennett, Jr., of the Virginia Commonwealth University in Richmond. First author Paula Keeney of the University of Virginia in Charlottesville used laser capture microdissection to collect individual spinal motor neurons, then the researchers analyzed their mtDNA content. The pair published their results online in Molecular Neurodegeneration on May 26. Stephen Ginsburg of the Nathan Kline Institute in Orangeburg, New York, who was not involved in the study, called the results highly preliminary, but provocative.

Weakened mitochondria are frequently linked to neurodegenerative disease. In the substantia nigra of tissue from people who had Parkinson disease, individual dopaminergic neurons often contain mtDNA with deletions (see ARF related news story on Bender et al., 2006 and Kraytsberg et al., 2006). Mitochondrial DNA also accumulates mutations and deletions with normal aging.

In spinal cord tissue from people who had ALS, cytochrome c oxidase activity is lower than normal, but that’s just one indicator of mitochondrial dysfunction (Borthwick et al., 1999; Fujita et al., 1996). Previous researchers have also found that mtDNA is mutated and diminished in copy number in ALS spinal cords (Wiedemann et al., 2002).

Bennett has a longstanding interest in mitochondria and neurodegeneration. He co-developed a mitochondria-targeted experimental drug that is currently going into Phase 3 trials by Knopp Neurosciences of Pittsburgh, Pennsylvania (KNS-760704; see ARF related drug story). In addition, a former member of his laboratory, Shaharyar Khan, invented a method to deliver fresh, whole mitochondrial genomes to damaged cells. Khan has since started the biotech company Gencia Corporation in Charlottesville to try to develop his idea. (Bennett has financial ties to Knopp’s drug, but not to Gencia.) Bennett hypothesized that since motor neurons are the specific cell type that degenerates in ALS, they might contain mtDNA deletions similar to those found in Parkinson disease.

Keeney collected individual anterior motor neurons from postmortem samples of 10 people who had sporadic ALS, plus seven age-matched, healthy spinal cords. In addition, she collected Purkinje neurons from unaffected brains to serve as another control sample. The researchers used quantitative PCR to determine copy number of the mitochondrial genes ND2, CO3, and ND4. The qPCR method Bennett and Keeney used is uncommon, Ginsberg said, compared to sequencing and microarrays that researchers usually employ. Others have used a similar approach, with qRT-PCR, to analyze glutamate receptor expression in single neurons (Heath et al., 2002), but this study is the first to target mtDNA.

Motor neurons from both ALS and control samples had widely varying copy numbers, ranging from zero to 300,000. This kind of genomic variability between individual cells is fairly common, Ginsberg said. He explained that some variability is usually due to experimental error, but some is a real representation of DNA levels. Keeney and Bennett also examined copy number in the Purkinje neurons and found it less variable than in motor neurons, suggesting that spinal motor neuron results are accurate, not only due to experimental variability.

Next, the researchers compared copy number of the individual genes they amplified. If the mitochondrial chromosome were whole, then the genes should be present in equal amounts. ND2 resides on a part of the chromosome less susceptible to deletion, so Bennett and Keeney calculated the ratio of CO3:ND2 and ND4:ND2 copy numbers. The answer was less than 1:1 in many cells. The scientists inferred, therefore, that ND4 and CO3 were deleted in some mitochondrial chromosomes in those cells. This was common in ALS spinal neurons, Bennett said, and slightly less common in control motor neurons. However, not all ALS cases showed evidence of many mitochondrial deletions.

The authors did not sequence their samples to determine the exact location of these inferred deletions. “Sequencing would be proof positive,” Ginsberg said. The researchers have shown that mitochondria are likely to malfunction in anterior spinal motor neurons, which could account for the mitochondrial problems in spinal cord homogenates. However, Ginsberg said, other motor neuron populations in the spinal cord could also contribute to the spinal cord’s mitochondrial malady, and should be tested individually as well.

The mechanism by which the mitochondrial chromosome falls apart in disease and normal aging is uncertain, Bennett said, but scientists suspect it happens when DNA damage interferes with replication (Krishnan et al., 2008). Reactive oxygen species increase with age, and mitochondrial DNA is particularly susceptible to the oxidative damage of individual bases. The cell may attempt to repair this damage by cutting out the altered nucleotide, but leave a single-strand break. During DNA replication, the mitochondrial DNA polymerase might “skip” the broken areas, “like a needle skipping tracks on a record,” Bennett said. The shortened chromosomes can then replicate faster, outcompeting full-size mitochondrial DNA (Fukui and Moraes, 2009).

Bennett acknowledged that the damaged mtDNA in ALS motor neurons may not cause the disease, but could be a downstream effect of degenerating nerves. And, Ginsberg noted, the Virginia researchers only analyzed neurons that survived; as in any postmortem study, these data do not tell what happened earlier in the disease as motor neurons began to degenerate. But regardless of whether the mutant mtDNA is cause or effect, the authors write, missing mitochondrial genes will impair neuron function, interfering with cellular calcium regulation and stress response. These “metabolically impotent” neurons could contribute to disease in some people with ALS, Bennett said.

If so, Khan’s mtDNA replacement therapy might be useful. The approach relies on the mitochondrial transcription factor A (TFAM), which tightly binds mtDNA. Khan’s recombinant protein allegedly carries replacement mtDNA throughout the body, crosses the blood-brain barrier, and enters the neuron to deliver the goods to mitochondria. Keeney and colleagues recently reported that TFAM delivers new mitochondrial genomes and improves respiration in a cybrid model of Parkinson disease (Keeney et al., 2009).

“If one can improve the bioenergetics inside a neuron, it can only be for the benefit of that particular neuron,” Bennett said. Ginsberg was more skeptical of the therapy, noting that with increased mitochondrial activity comes increased production of ROS. These might damage cells further, he speculated, if the cell is ill equipped to deal with them. Bennett told ARF in an e-mail that although increased ROS is a potential side effect, the researchers have not observed it in rodent studies.

Overall, Ginsberg felt these initial results were worthy of further inquiry. For one, he suggested that scientists compare mitochondrial genomes in sporadic versus familial ALS. In addition, researchers can look for similar mtDNA defects in mouse models of ALS. If these defects are present in animals, Bennett wants to try the TFAM therapy in ALS mice.—Amber Dance